Secondary Glycosylation Research Articles

The localization and timing of post-translational modifications of rat rhodopsin

Rat retinas were labeled by incubation with, or intravitreal injection of, [ 14C]leucine along with tritiated palmitic acid, glucosamine or galactose. At selected intervals, subcellular fractions were prepared on linear sucrose gradients and rhodopsin was extracted and purified by affinity chromatography and gel electrophoresis. Time courses revealed that leucine rapidly and transiently labeled the rhodopsin in the rough endoplasmic reticulum (RER), with a maximum at 1·5 hr post-injection. Subsequently, the rod outer segments (ROS) contained the labeled rhodopsin, with the ROS labeling maximally at 6–12 hr. Palmitate labeling followed the same pattern but was subject to a delay, presumably because of a large intracellular pool of the fatty acid. With palmitate the RER rhodopsin was not maximally labeled until 12 hr. The acylation of rhodopsin takes place in the RER sometime after the polypeptide has been translated but before transport to the Golgi. Glucosamine labeling was also delayed because of intracellular pools of the sugar or its metabolic derivatives. But because of secondary glycosylation in the Golgi, the rhodopsin in the ROS also labeled maximally with glucosamine at about 6 hr. Administration of [ 3H]galactose resulted in the labeling of rhodopsin both in vivo and in vitro, in part possibly because of its conversion to mannose and subsequent insertion into the core oligosaccharide on the RER. However, in the ROS the ratio of tritium, derived from [ 3H]galactose, to [ 14C]leucine decreased by a factor of 2 between 6 and 24 hr post-injection. Moreover, between 6 and 12 hr post-injection, labeled rhodopsin molecules in the ROS underwent a shift in mobility on gels indicative of trimming to a lower molecular weight. Thus some sugar residues may be added to the rhodopsin in the inner segment and removed in the ROS.

Biosynthesis and processing of human immunodeficiency virus type 1 envelope glycoproteins: effects of monensin on glycosylation and transport

When human immunodeficiency virus type 1 envelope glycoproteins were expressed in 293 cells by using a recombinant adenovirus expression vector, the envelope precursor (gp160) was initially glycosylated by cotranslational addition of N-linked high-mannose oligosaccharide units to the protein backbone and then cleaved to gp120 and gp41. The subunits gp120 and gp41 were then further modified by the addition of fucose, galactose, and sialic acid, resulting in glycoproteins containing a mixture of hybrid and complex oligosaccharide side chains. A fraction of glycosylated gp160 that escaped cleavage was further modified by the terminal addition of fucose and galactose, but the addition of sialic acid did not occur, consistent with the notion that it is compartmentalized separately from the gp120 envelope protein. Processing and transport of gp160 were blocked by the monovalent ionophore monensin, which at high concentrations (25 microM and above) was a potent inhibitor of the endoproteolytic cleavage of gp160; at lower concentrations (1 to 10 microM), it selectively blocked the secondary glycosylation steps so that smaller products were produced. Monensin (1 microM) treatment also resulted in a reduction in syncytium formation, which was observed when recombinant infected cells were cocultivated with CD4-bearing HeLa cells. The infectivity of human immunodeficiency virus type 1 was also reduced by monensin treatment, a decrease that may be due to incompletely glycosylated forms of gp120 that have a lower affinity for the CD4 receptor.

102] Glycosylation of rhodopsin

This chapter discusses the glycosylation of rhodopsin. The oligosaccharide of rhodopsin is relatively simple, consisting of two separate chains, each composed of mannosc and N -acetylglucosa-mine. Structures of this type are synthesized on microsomal membranes as lipid-linked oligosaccharides which are transferred en bloc to an asparagine residue of the newly synthesized polypeptide. The transfer of galactose and fucose from the respective sugar nucleotides to rhodopsin can be demonstrated with bovine rod outer segment preparations. The site of this secondary glycosylation of opsin can be demonstrated by the use of another inhibitor in vitro . A large intracellular pool of uridine diphosphate (UDP)-N-acetylglucosamine is inferred from the relatively slow autoradio-graphic labeling of the rough endoplasmic reticulum. The synthesis of lipid-linked oligosaccharide intermediates has been described in chick embryo retina. Comparable oligosaccharide-lipids are formed by cell-free bovine retina preparations with evidence that they are also transferred to rhodopsin.

Structure and processing of the mouse mammary tumor virus glycoprotein precursor pr73env.

The polyprotein precursor to the envelope glycoproteins of mouse mammary tumor virus was investigated by using subcellular fractionation procedures, pactomycin mapping techniques, tunicamycin inhibition of glycosylation, and endo-beta-N-acetyl glucosaminidase H-catalyzed removal of glycosylated residues in order to characterize the biosynthesis and processing of the precursor. The results suggest that the precursor (Pr73env) is synthesized on the rough endoplasmic reticulum as a transmembrane protein, with the carboxyl terminus remaining on the cytoplasmic side. The apoprotein as an estimated molecular weight of 60,000 and acquires five core oligosaccharide units during synthesis. Cleavage of the precursor precedes the secondary glycosylation steps and therefore probably occurs before transport to the plasma membrane. However, a minor population of Pr73env containing complex oligosaccharides was also found in the plasma membrane. The order of the glycoproteins in the precursor, as determined by pactomycin mapping, in NH2-gp52-gp36-COOH.

Differential effects of puromycin on the incorporation of precursors of rhodopsin in bovine retina.

Bovine retinas incubated in vitro sustain the synthesis of opsin and rhodopsin as monitored by the incorporation of labeled leucine, mannose, and glucosamine. Puromycin, an inhibitor of protein synthesis, effectively blocks the incorporation of leucine and mannose into opsin and rhodopsin of rod outer segments. However, the incorporation of glucosamine into opsin and rhodopsin is not immediately blocked. Instead, it continues for a time suggesting not only core oligosaccharide synthesis but also the secondary glycosylation of a pool of preformed opsin which is thought to be transiently accumulated in the photoreceptor Golgi complex. Galactose, not normally found in rhodopsin, is also incorporated into both opsin and rhodopsin. This incorporation appears to be completely insensitive to puromycin, suggesting that it may occur in the rod outer segments involving only preexisting glycoproteins.